Primary immunodeficiency diseases provide unique opportunities to study the role of discrete cells and molecules in the immune response. Our general aim is to understand the role of CD40 and of its ligand in immune function through the study of patients and of genetically engineered mice with CD40l and CD40 deficiency. CD40 is expressed on B cells, thymic epithelium and dendritic cells and plays an important role in B cell survival, activation, differentiation and class switching. The ligand for CD40 (CD40L/gp39) is expressed exclusively on activated T cells in a developmentally regulated manner. We have recently found that mutations in the CD40L gene resulting in loss of function or expression of CD40L are the basis for the X-linked HyperIgM syndrome(HIGMX-1). Deficiency in CD40 has not yet been described. In Aim 1, we propose to analyze in detail the mechanisms of CD40L deficiency in HIGMX-1. We will characterize the defect in the CD40L gene in a panel of HIGMX-1 patients at the cDNA level and at the genomic level. The latter requires an analysis of the genomic organization and of the minimal transcriptional unit of the human CD40L gene. In Aim 2, we propose to construct a murine model of HIGMX-1 to gain a better understanding of CD40L deficiency and to begin testing novel therapeutic modalities for HIGMX-1. To this purpose, we will generate CD40L deficient mice by RAG-2-deficient blastocyst complementation and by disruption of the CD40L germline gene. We will also construct transgenic mice with discrete mutations in CD40L to mimic the mutations in HIGMX-1 patients and to define the function of CD40L domains. In Aim 3, we will examine the role of CD40 in the development of immune cells and define the phenotype of CD40 deficiency through the study of CD$0 knockout mice. The role of CD40 in B cell development and function will be defined by examining CD40 deficient mice constructed by RAG-2 deficient blastocyst complementation. The role of CD40 in the development and function of thymic epithelial cells and dendritic cells will be analyzed in mice with disrupted CD40L germline gene.